Summary of Work: Mapping epitopes of the Human Immunodeficiency Virus (HIV) is important for the diagnosis of infection and for the development of vaccines and therapeutics for Acquired Immune Deficiency Syndrome (AIDS). We have been probing epitopes on the HIV proteins gp140, gp120, and p24. The initial step in the entry of HIV into the host cell is binding of a trimer of the envelope glycoprotein gp120 and associated gp41 to the cellular receptor CD4. Also gp120 and gp41 elicit the major components of the protective immune response against HIV in humans and chimpanzees. gp120, gp41 and synthetic peptides containing sequences of these proteins have been investigated as potential vaccine candidates. Similarly, HIV p24 elicits the first antibodies upon HIV infection. As the HIV infection progresses to AIDS, there is a simultaneous reduction in anti-p24 antibody titer. It has been proposed that a combination vaccine eliciting antibodies to both gp120 and p24 may be useful in combating HIV infection. Thus, knowledge of the antigenic determinants on p24 and gp120, especially those eliciting the formation of protective antibodies, is extremely important in the development of a vaccine. We have combined proteolytic footprinting and MALDI/MS to map epitopes on the native proteins recognized by antibodies. In this method, proteins affinity-bound to an immobilized antibody are proteolytically cleaved and the unbound fragments are removed by washing. The bound fragments containing the epitope are characterized by directly analyzing the immobilized antibody by MALDI/MS. We are currently mapping epitopes on gp120, p24 and gp41 recognized by human MAbs isolated from sera of HIV infected individuals. The anti-HIV gp41 MAb, 2F5, is one of a limited number (three) of antibodies that have been found to be broadly neutralizing in vivo. Using traditional methods for defining epitopes, a short 6 amino acid sequence on gp41 was determined to be the antigen. All attempts, however, to use this sequence to induce neutralizing antibodies have failed. Antibodies are readily formed against the peptide, but they Abs do not have neutralizing properties. The hypothesis for our experiments is that the epitope may be conformational or discontinuous with some residues from gp120 being involved. We investigated the nature of the 2F5 epitope on a recombinant disulfide stabilized glycoprotein gp140. The disulfide bond stabilizes the association of gp120 and gp41ectodomains as observed during viral infection. We determined that the functional epitope recognized by the neutralizing MAb is significantly longer in length, 15-16 residues, but does not include residues from the gp120 portion. We are currently working on the determination of a second in vivo neutralizing epitope, 4E10, putatively recognizing a gp41 sequence. We are also determining conformational epitopes on the p24 capsid protein to determine the mechanism of how certain human anti-p24 MAbs block p24 antigen presentation to CD4 T helper cells while others do not.